Abstract
Introduction
Root canal treatment is incomplete without usage of intra canal medicaments. They help in the reduction of bacterial count and its by-products, making canals clean and decreasing postoperative pains.
Aim
The aim of this study was to evaluate and compare the antimicrobial activity of triple antibiotic paste, Moxifloxacin, calcium hydroxide and 2% Chlorhexidine (CHX) gel in elimination of Enterococcous faecalis (E. faecalis).
Materials and Methods
Seventy-five root blocks were obtained from extracted single rooted human teeth. The canal diameter was increased using Gates- Glidden drill up to size 3 and then contaminated with E. faecalis for 21 days. The contaminated samples were then divided into following 5 groups. Group 1: Saline (negative group), Group 2: Calcium hydroxide Ca(OH)2, Group 3: 2% CHX gel, Group 4: Triple Antibiotic Paste (TAP) (50 μg – metronidazole of 400 mg, 50 μg – minocycline of 100 mg, 50 μg – ciprofloxacin of 100 mg) and Group 5: Moxifloxacin (50 μg – moxifloxacin of 400 mg). Dentin debris was obtained at the end of first, 7th, and 10th day using Gates Glidden drill sizes 4 and 5. The bacterial load was assessed by counting the number of Colony Forming Units (CFUs). The data were analyzed with the ANOVA and Post-Hoc tests to assess the differences in antibacterial efficacy between groups (p=<0.001).
Results
A 2% CHX gel alone completely inhibited the growth of E. faecalis after one, seven and 10 days. The 2% CHX gel was the most effective medicament against E. faecalis, as it showed significant differences with normal saline, calcium hydroxide, Moxifloxacin or triple antibiotic paste at all time intervals. The triple antibiotic paste group showed a moderate antibacterial effect as its difference with all group was significantly better at all days. Moxifloxacin was more effective than calcium hydroxide on 7th and 10th day.
Conclusion
Best antimicrobial efficacy was shown by 2% CHX gel. Moxifloxacin was equally efficient compared to triple antibiotic paste against E. faecalis at longer intervals of time.
Keywords: Antimicrobial efficacy, Dentinal tubule, Fluoroquinolone, Intra canal medicament, Root canal treatment
Introduction
Microorganisms play an important role in pulpal and periapical diseases and their elimination during endodontic treatment is essential for success of treatment [1]. The infection in the root canal system is poly microbial in nature which is dominated by anaerobes [2]. Primary root canal infection involves various bacterial species which is dominated differentially at different stages of infection [3]. The major cause of failure of root canal treatment is the persistence of microorganisms in the apical part of the root canal. Most of the microorganisms such as enterococci and streptococci are capable of surviving different conditions of filled root canals [4]. In most of the failed cases of root canal treatment teeth a Gram +ve facultative anaerobe E. faecalis is the frequently isolated microorganism [5]. It’s because of its unique capability to invade dentinal tubules depth and surviving extreme periods of pH and limited nutrition [6]. The placement of intracanal medicaments between the appointments helps in eradication of the surviving microorganism and promoting the successful outcome of the treatment [7].
Antimicrobial irrigants and medicaments are used during instrumentation in canal which reduce bacterial population effectively however some bacteria may survive in lateral and accessory root canal, isthmi and apical delta [4,8]. The most common intracanal medicament in root canal treatment is calcium hydroxide which has it’s effect on most of the bacteria because of its high pH. E. faecalis does not responds to Ca(OH)2 as it resides deeper part of dentinal tubules where pH seen to be stable because of buffering action of dentin [9]. CHX is widely used in endodontics due to its antimicrobial activity. It’s used as both irrigant and intracanal medicament [10] due to its inhibitory effect on bacteria which is found in endodontic infection against Gram positive and gram negative microorganisms [11]. The efficacy of CHX is because of positively charged CHX molecule which interacts with negatively charged microbial cell wall and thereby, increasing the permeability of the cell wall and precipitation of cytoplasmic contents resulting in cell death [12].
Triple antibiotic paste is a routinely used intracanal medicament during regenerative endodontics practice. It contains mixture of ciprofloxacin, metronidazole and tetracycline derivative called minocycline. The action of this mixture against various endodontics microbes had been studied extensively and proved to be beneficial [13].
Moxifloxacin is a new fluoroquinolone with expended spectrum of activity, including anaerobes and Gram positive organisms, especially the multi-resistant ones [14]. It has been found to be one of the most active antibiotics against E. faecalis with lowest MIC50 and MIC90 [15].
The laboratory study was carried out to test the efficacy of triple antibiotic paste, Moxifloxacin, calcium hydroxide and chlorhexidine in elimination of E. faecalis.
Materials and Methods
This study was conducted at the Department of Conservative and Endodontics in Collaboration with Department of Microbiology at Rungta College of Dental Science and Research Bhilai, Chhattisgarh, India.
The model previously used by Haapasalo and ørstavik was modified for this study [16].
Cleaning and Disinfection Procedure
Seventy-five (n=75) freshly extracted human teeth from department of Oral and Maxillofacial Surgery were selected. Teeth were stored in 10% formalin for 24 hours for disinfection and organic tissue fixation was used. Afterwards, these teeth were cleaned with the help of saline and middle third of each root were obtained with the help of diamond disk. Single rooted single canal teeth, closed apex, straight root were included in the study. Teeth with curved root, calcified root, double root canal and caries teeth were excluded.
Preparation of Block
The middle third of each root canal were obtained by cutting 6 mm slices from coronal and apical parts of the roots. The internal diameter of blocks was standardized with the help of Gates-Glidden drill sizes 1, 2, and 3 were used to enlarge the canal. The blocks were submerged in 17% EDTA followed by 2.5% sodium hypochlorite for 5 minutes to remove organic and inorganic debris. In order to remove the residual chemicals, the blocks were dipped in ultrasonic cleaner for 5 minutes and were autoclaved afterwards at 1210C for 30 minutes then they were immersed in 1 ml of Tryptic Soy broth (TS broth) to allow better penetration of broth into dental tubules.
Contamination of Block
Culture of pure E. faecalis was used in this study. Each block was contaminated with micro centrifuge tube containing 1ml of TS broth and 50 μL of inoculum of E. faecalis. During a period of three weeks every second day each block were transferred to fresh TS broth containing E. faecalis. The blocks were removed irrigated with 5 ml of sterile saline at the end of incubation period. The sample were than assigned to groups (n=15) as shown in [Table/Fig-1]. The triple antibiotic paste, calcium hydroxide powder, and Moxifloxacin were mixed in paste like consistency using distilled water. The 2% CHX gel and other intracanal medicaments were carried into canal using lentulo spirals. After removal of excess material, the coronal and apical part sealed with wax than blocks were incubated with at 370C and 100% humidity.
[Table/Fig-1].
Study groups included in the research.
| Group 1 | Saline (negative group) |
| Group 2 | Calcium hydroxide powder |
| Group 3 | 2% CHX gel |
| Group 4 | Triple antibiotic paste 50 μg - metronidazole of 400 mg 50 μg - minocycline of 100 mg 50 μg - ciprofloxacin of 100 mg |
| Group 5 | Moxifloxacin 50 μg - moxifloxacin of 400 mg |
Antimicrobial Assessment
Antimicrobial assessment was carried out at end of 1st, 7th, 10th day. Ten blocks from each group at different incubation period were taken. After ending of each incubation period the wax was removed from the root end and irrigated with 10 ml of sterile saline and subsequently dried with paper points. Gates Glidden drill size 4-5 were used to collect dentinal debris in laminar hood flow and they were transferred to micro centrifugal having 1 ml of sterile TS broth then subsequently incubated in anaerobic environment at 37°C for 24 hours. After the ending of anaerobic incubation cycle the content of each tube was serially diluted for 3 times in 100 ml of broth in 900 ml of sterile broth and at last 100 ml of this diluted sample was spread over TS broth agar and allowed to incubated for 48 hours. Colonies were counted and tabulated.
Statistical Analysis
ANOVA and Post-Hoc test were used to compare the difference in antimicrobial efficacy between all groups (p=<0.001).
Results
The results are sequentially shown in [Table/Fig-2,3,4,5,6 and 7]. The saline group (negative) showed larger infiltration in dentinal tubules with E. faecalis colonies confirming the infection in all blocks. The 2% CHX gel was the most effective medicament against E. faecalis, as it showed significant differences with saline, calcium hydroxide, Moxifloxacin or triple antibiotic paste in all time intervals.
[Table/Fig-2].
Comparison of mean number of E. faecalis colony count (10-2) among various intracanal medicaments on 1st day.
| Moxifloxocin | TAP | Calcium Hydroxide | Chlor hexidine | Normal Saline | |
|---|---|---|---|---|---|
| Mean | 2.72 | 2.16 | 2.48 | 1.22 | 4.72 |
| Standard Deviation | 0.0836 | 0.0894 | 0.1303 | 0.1643 | 0.1095 |
F-value= 581.2, p-value= <0.001
[Table/Fig-3].
Comparison of mean number of E. faecalis colony count (10-2) among various intracanal medicaments on 1st day.
| Moxifloxocin | TAP | Calcium Hydroxide | Chlorhexidine | normal Saline | |
|---|---|---|---|---|---|
| Mean | 2.26 | 1.72 | 2.28 | 0.9 | 5.32 |
| Standard Deviation | 0.0894 | 0.1095 | 0.1303 | 0.1414 | 0.1095 |
F-value= 1016.9, p-value= <0.001
[Table/Fig-4].
Comparison of mean number of E. faecalis colony count (10-2) among various intracanal medicaments on 10th day.
| Moxifloxocin | TAP | Calcium hydroxide | Chlorhexidine | Normal Saline | |
|---|---|---|---|---|---|
| Mean | 2.46 | 2.46 | 2.36 | 1.36 | 8.5 |
| Standard Deviation | 0.0547 | 0.08944 | 0.0547 | 0.2190 | 0.1000 |
F-value= 2866, p-value= <0.001
[Table/Fig-5].
Intergroup comparison among various intracanal medicaments on 1st day using Post-Hoc test.
| Duration | Intracanal Medicament | Comparison with Standard intracanal Medicaments | p-value |
|---|---|---|---|
| On 1st day | Moxifloxacin | Triple Antibiotic Paste | <0.001 |
| Calcium Hydroxide | 0.034 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Triple Antibiotic Paste | Moxifloxacin | <0.001 | |
| Calcium Hydroxide | 0.003 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Calcium Hydroxide | Moxifloxacin | 0.034 | |
| Triple Antibiotic Paste | 0.003 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Chlorhexidine | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Normal Saline | <0.001 | ||
| Normal Saline | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Chlorhexidine | <0.001 |
[Table/Fig-6].
Intergroup comparison among various intracanal medicaments on 7th day using Post-Hoc test.
| Duration | Intracanal Médicament | Comparison with Standard intracanal Medicaments | p-value |
|---|---|---|---|
| On 7 th day | Moxifloxacin | Triple Antibiotic Paste | <0.001 |
| Calcium Hydroxide | 0.999 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Triple Antibiotic Paste | Moxifloxacin | <0.001 | |
| Calcium Hydroxide | <0.001 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Calcium Hydroxide | Moxifloxacin | 0.999 | |
| Triple Antibiotic Paste | <0.001 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Chlorhexidine | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Normal Saline | <0.001 | ||
| Normal Saline | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Chlorhexidine | <0.001 |
[Table/Fig-7].
Intergroup comparison among various intracanal medicaments on 10th day using Post-Hoc test.
| Duration | Intracanal Medicament | Comparison with Standard intracanal medicaments | p-value |
|---|---|---|---|
| On 10th day | Moxifloxacin | Triple Antibiotic Paste | 1.000 |
| Calcium Hydroxide | 0.684 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Triple Antibiotic Paste | Moxifloxacin | 1.000 | |
| Calcium Hydroxide | 0.684 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Calcium Hydroxide | Moxifloxacin | 0.684 | |
| Triple Antibiotic Paste | 0.684 | ||
| Chlorhexidine | <0.001 | ||
| Normal Saline | <0.001 | ||
| Chlorhexidine | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Normal Saline | <0.001 | ||
| Normal Saline | Moxifloxacin | <0.001 | |
| Triple Antibiotic Paste | <0.001 | ||
| Calcium Hydroxide | <0.001 | ||
| Chlorhexidine | <0.001 |
The triple antibiotic paste group showed a moderate antibacterial effect as its difference with all group was significant in all days. Moxifloxacin was effective against calcium hydroxide in 7th and 10th day.
Discussion
Various studies models are proposed for evaluation of different intracanal medicament against the resistant microflora in endodontics [8,16]. The methodology for evaluating the efficacy of intra canal medicament was adopted from the study done by Adl A et al., [17]. E. faecalis was selected for this study as its most commonly associated with failed root canal treated teeth [18].
The triple antibiotic paste, is a mixture of metronidazole, ciprofloxacin, and minocycline which is used as an intracanal medicament for disinfection of immature necrotic teeth, during regenerative endodontic procedures. Moxifloxacin is a synthetic fluoroquinolone of 4th generation which differ from earlier class of fluoroquinolones such as levofloxacin, ciprofloxacin having greater potential against gram positive and gram negative bacteria [19]. It has excellent bioavailability, a long half-life, and good tissue penetration [20]. CHX is commonly used as irrigant and intracanal medicament in routine endodontic procedure. It is effective against wide variety of gram positive and negative bacteria, fungus and yeasts.
Tankovic J et al., found moxifloxacin to be 2-4 times much more active than sparfloxacin, ciprofloxacin and 16 times more active than ofloxacin [21]. According to Edmiston CE et al., moxifloxacin was proved to be highly efficient against methicillin susceptible S. aureus, E. faecalis and streptococcus when compared with fluoroquinolones [22]. Pinheiro E et al., demonstrated high (100%) susceptibility of all the 21 isolates of E. faecalis to amoxicillin, clavulanic acid, moxifloxacin and vancomycin while ciprofloxacin demonstrated less susceptibility (80%) compound to the mentioned drugs [23]. Schubert S and Dalhoff A concluded the activity of moxifloxacin to be more effective than imipenem and ertapenem against E. faecalis in monoculture and mixed culture [24]. Al Ahmad A et al., confirmed the susceptibility of all strains of E. faecalis for moxifloxacin [25]. In present study, moxifloxacin was found to be less effective than triple antibiotic paste on 1st and 7th day but on 10th day the activity was comparable to triple antibiotic paste. This may be attributed because of its wider spectrum against both Gram positive and negative organisms and different mechanism of action than ciprofloxacin.
In a study, by Lindskog S et al., 2% CHX gel was active against E. faecalis even after 21 days of root dentin treatment [26]. Dametto et al., confirmed that 2% CHX gel and 2% CHX liquid significantly reduced the number of E. faecalis colonies. [27]. The most effective medicament in present study against E. faecalis was found to be CHX as it shows sustained activity against E. faecalis at different time intervals. This can be explained by contact between molecules of positive charge and phosphate group of negative charge allowing CHX molecules to enter bacterial cell wall with toxic effect. The present study showed no considerable effect of CHX on E. faecalis which was confirmed with previous studies about the resistance of E. faecalis against CHX [27]. E. faecalis is resistant to calcium hydroxide, especially when a high pH is not maintained [28]. This may be due to fact that, E. faecalis can survive a pH of 11 and that level of pH couldn’t be reduced because of the buffering capacity of dentin. The resistant to pH change might also be due to the blockage of dentinal tubules by the layer of number of bacteria used in this study [29].
In the present study, triple antibiotic paste is more effective than CHX which coincides with the results of the study done by Madhubala MM et al., in which propolis and triple antibiotic paste showed higher antibacterial effects than CHX on E. faecalis [30]. A recent in vitro study showed that compared to CHX, the triple antibiotic paste is highly effective against E. faecalis. In the study done by Adl A et al., triple antibiotic paste, including metronidazole, ciprofloxacin and minocycline, had an appropriate effect on E. faecalis [31].
As our study was limited to the number of samples and the medicaments, more studies are needed with greater sample size for evaluating the efficacy of moxifloxacin on E. faecalis as an intracanal medicament in failed root canal treated cases. In coming future, we may find moxifloxacin as single drug replacement for the commonly used intracanal medicaments triple antibiotic paste and CHX, in resistant microbes of root canals.
Conclusion
Within the perceptive of the study it can be concluded that 2% CHX gel is the most effective medicament against E. faecalis in infected root canals.
Acknowledgments
We thank Dr Reena Kulshreshtha, Reader, Department of Microbiology, RCDSR for her guidance throughout the study.
Financial or Other Competing Interests
None.
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